title: Förster resonance energy transfer microscopy and spectroscopy for localizing protein-protein interactions in living cells.
abstract: The fundamental theory of Förster resonance energy transfer (FRET) was established in the 1940s. Its great power was only realized in the past 20 years after different techniques were developed and applied to biological experiments. This success was made possible by the availability of suitable fluorescent probes, advanced optics, detectors, microscopy instrumentation, and analytical tools. Combined with state-of-the-art microscopy and spectroscopy, FRET imaging allows scientists to study a variety of phenomena that produce changes in molecular proximity, thereby leading to many significant findings in the life sciences. In this review, we outline various FRET imaging techniques and their strengths and limitations; we also provide a biological model to demonstrate how to investigate protein-protein interactions in living cells using both intensity- and fluorescence lifetime-based FRET microscopy methods.
authors.affiliation: The W.M. Keck Center for Cellular Imaging (KCCI), Department of Biology, Physical and Life Sciences Building, University of Virginia, Charlottesville, Virginia.
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Sun
authors.forename: Christina
authors.initials: C
authors.lastname: Rombola
authors.forename: Vinod
authors.initials: V
authors.lastname: Jyothikumar
authors.forename: Ammasi
authors.initials: A
authors.lastname: Periasamy
mesh_terms: D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D025941:Protein Interaction Mapping / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000737:chemistry; D059010:Single-Cell Analysis / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins
keywords: FLIM; FLIM-FRET; FRET; protein-protein interactions